The invention concerns functional chimeras between CD4 fragments and immune cell receptors which are capable of directing immune cells to lyse HIV-infected cells, but which do not render the immune cells susceptible to HIV infection. The invention therefore provides a novel and effective HIV therapeutic.
T cell recognition of antigen through the T cell receptor is the basis of a range of immunological phenomena. The T cells direct what is called cell-mediated immunity. This involves the destruction by cells of the immune system of foreign tissues or infected cells. A variety of T cells exist, including xe2x80x9chelperxe2x80x9d and xe2x80x9csuppressorxe2x80x9d cells, which modulate the immune response, and cytotoxic (or xe2x80x9ckillerxe2x80x9d) cells, which can kill abnormal cells directly.
A T cell that recognizes and binds a unique antigen displayed on the surface of another cell becomes activated; it can then multiply, and, if it is a cytotoxic cell, it can kill the bound cell. HIV and Immunopathogenesis
In 1984 HIV was shown to be the etiologic agent of AIDS. Since that time the definition of AIDS has been revised a number of times with regard to what criteria should be included in the diagnosis. However, despite the fluctuation in diagnostic parameters, the simple common denominator of AIDS is the infection with HIV and subsequent development of persistent constitutional symptoms and AIDS-defining diseases such as a secondary infections, neoplasms, and neurologic disease. Harrison""s Principles of Internal Medicine, 12th ed., McGraw Hill (1991).
HIV is a human retrovirus of the lentivirus group. The four recognized human retroviruses belong to two distinct groups: the human T lymphotropic (or leukemia) retroviruses, HTLV-1 and HTLV-2, and the human immunodeficiency viruses, HIV-1 and HIV-2. The former are transforming viruses whereas the latter are cytopathic viruses.
HIV-1 has been identified as the most common cause of AIDS throughout the world. Sequence homology between HIV-2 and HIV-1 is about 40% with HIV-2 being more closely related to some members of a group of simian immunodeficiency viruses (SIV). See Curran et al., Science 329:1357-1359 (1985); Weiss et al., Nature 324:572-575 (1986).
HIV has the usual retroviral genes (env, gag, and pol) as well as six extra genes involved in the replication and other biologic activities of the virus. As stated previously, the common denominator of AIDS is a profound immunosuppression, predominantly of cell-mediated immunity. This immune suppression leads to a variety of opportunistic diseases, particularly certain infections and neoplasms.
The main cause of the immune defect in AIDS has been identified as a quantitative and qualitative deficiency in the subset of thymus-derived (T) lymphocytes, the T4 population. This subset of cells is defined phenotypically by the presence of the CD4 surface molecule, which has been demonstrated to be the cellular receptor for HIV. Dalgleish et al., Nature 312:763 (1984). Although the T4 cell is the major cell type infected with HIV, essentially any human cell that expresses the CD4 molecule on its surface is capable of binding to and being infected with HIV.
Traditionally, CD4+ T cells have been assigned the role of helper/inducer, indicating their function in providing an activating signal to B cells, or inducing T lymphocytes bearing the reciprocal CD8 marker to become cytotoxic/suppressor cells. Reinherz and Schlossman, Cell 19:821-827 (1980); Goldstein et al., Immunol. Rev. 68:5-42 (1982).
HIV binds specifically and with high affinity, via a stretch of amino acids in the viral envelope (gp120), to a portion of the V1 region of the CD4 molecule located near its N-terminus. Following binding, the virus fuses with the target cell membrane and is internalized. Once internalized it uses the enzyme reverse transcriptase to transcribe its genomic RNA to DNA, which is integrated into the cellular DNA where it exists for the life of the cell as a xe2x80x9cprovirus.xe2x80x9d
The provirus may remain latent or be activated to transcribe MRNA and genomic RNA, leading to protein synthesis, assembly, new virion formation, and budding of virus from the cell surface. Although the precise mechanism by which the virus induces cell death has not been established, it is believed that the major mechanism is massive viral budding from the cell surface, leading to disruption of the plasma membrane and resulting osmotic disequilibrium.
During the course of the infection, the host organism develops antibodies against viral proteins, including the major envelope glycoproteins gp120 and gp41. Despite this humoral immunity, the disease progresses, resulting in a lethal immunosuppression characterized by multiple opportunistic infections, parasitemia, dementia, and death. The failure of the host anti-viral antibodies to arrest the progression of the disease represents one of the most vexing and alarming aspects of the infection, and augurs poorly for vaccination efforts based upon conventional approaches.
Two factors may play a role in the efficacy of the humoral response to immunodeficiency viruses. First, like other RNA viruses (and like retroviruses in particular), the immunodeficiency viruses show a high mutation rate in response to host immune surveillance. Second, the envelope glycoproteins themselves are heavily glycosylated molecules presenting few epitopes suitable for high affinity antibody binding. The poorly antigenic target which the viral envelope presents allows the host little opportunity for restricting viral infection by specific antibody production.
Cells infected by the HIV virus express the gp120 glycoprotein on their surface. Gp120 mediates fusion events among CD4+ cells via a reaction similar to that by which the virus enters the uninfected cells, leading to the formation of short-lived multinucleated giant cells. Syncytium formation is dependent on a direct interaction of the gp120 envelope glycoprotein with the CD4 protein. Dalgleish et al., supra; Klatzman et al., Nature 312:763 (1984); McDougal et al., Science 231:382 (1986); Sodroski et al., Nature 322:470 (1986); Lifson et al., Nature 323:725 (1986); Sodroski et al., Nature 321:412 (1986).
Evidence that the CD4-gp120 binding is responsible for viral infection of cells bearing the CD4 antigen includes the finding that a specific complex is formed between gp120 and CD4 (McDougal et al., supra). Other investigators have shown that the cell lines, which were non-infective for HIV, were converted to infectable cell lines following transfection and expression of the human CD4 cDNA gene. Maddon et al., Cell 46:333-348 (1986).
Therapeutic programs based on soluble CD4 as a passive agent to interfere with viral adsorption and syncytium-mediated cellular transmission have been proposed and successfully demonstrated in vitro by a number of groups (Deen et al., Nature 331:82-84 (1988); Fisher et al., Nature 331:76-78 (1988); Hussey et al., Nature 331:78-81 (1988); Smith et al., Science 238:1704-1707 (1987); Traunecker et al., Nature 331:84-86 (1988)); and CD4 immunoglobulin fusion proteins with extended half-lives and modest biological activity have subsequently been developed (Capon et al., Nature 337:525-531 (1989); Traunecker et al. Nature 339, 68-70 (1989); Byrn et al., Nature 344:667-670 (1990); Zettlmeissl et al., DNA Cell Biol. 9:347-353 (1990)). Although CD4 immunotoxin conjugates or fusion proteins show potent cytotoxicity for infected cells in vitro (Chaudhary et al., Nature 335:369-372 (1988); Till et al., Science 242:1166-1168 (1988)), the latency of the immunodeficiency syndrome makes it unlikely that any single-treatment therapy will be effective in eliminating viral burden, and the antigenicity of foreign fusion proteins is likely to limit their acceptability in treatments requiring repetitive dosing. Trials with monkeys affected with SIV have shown that soluble CD4, if administered to animals without marked CD4 cytopenia, can reduce SIV titer and improve in vitro measures of myeloid potential (Watanabe et al., Nature 337:267-270 (1989)). However a prompt viral reemergence was observed after treatment was discontinued, suggesting that lifelong administration might be necessary to prevent progressive immune system debilitation.
Cell surface expression of the most abundant form of the T cell antigen receptor (TCR) requires the coexpression of at least 6 distinct polypeptide chains (Weiss et al., J. Exp. Med. 160:1284-1299 (1984); Orloffhashi et al., Nature 316:606-609 (1985); Berkhout et al., J. Biol. Chem. 263:8528-8536 (1988); Sussman et al., Cell 52:85-95 (1988)), the xcex1/xcex2 antigen binding chains, the three polypeptides of the CD3 complex, and xcex6. If any of the chains are absent, stable expression of the remaining members of the complex does not ensue. xcex6 is the limiting polypeptide for surface expression of the complete complex (Sussman et al., Cell 52:85-95 (1988)) and is thought to mediate at least a fraction of the cellular activation programs triggered by receptor recognition of ligand (Weissman et al., EMBO J. 8:3651-3656 (1989); Frank et al., Science 249:174-177 (1990)). A 32 kDa type I integral membrane homodimer, xcex6 (zeta) has a 9 residue extracellular domain with no sites for N-linked glycan addition, and a 112 residue (mouse) or 113 residue (human) intracellular domain (Weissman et al., Science 238:1018-1020 (1988); Weissman et al., Proc. Natl. Acad. Sci. USA 85:9709-9713 (1988)). An isoform of xcex6 called xcex7 (eta) (Baniyash et al., J. Biol. Chem. 263:9874-9878 (1988); Orloff et al., J. Biol. Chem. 264:14812-14817 (1989)), which arises from an alternate mRNA splicing pathway (Jin et al., Proc. Natl. Acad. Sci. USA 87:3319-3233 (1990)), is present in reduced amounts in cells expressing the antigen receptor. xcex6xe2x88x92xcex7 heterodimers are thought to mediate the formation of inositol phosphates, as well as the receptor-initiated programmed cell death called apoptosis (Merxc4x87ep et al., Science 242:571-574 (1988); Merxc4x87ep et al., Science 246:1162-1165 (1989)).
Like xcex6 and xcex7, the Fc receptor-associated xcex3 (gamma) chain is expressed in cell surface complexes with additional polypeptides, some of which mediate ligand recognition, and others of which have undefined function. xcex3 bears a homodimeric structure and overall organization very similar to that of xcex6 and is a component of both the mast cell/basophil high affinity IgE receptor, Fcxcex5RI, which consists of at least three distinct polypeptide chains (Blank et al., Nature 337:187-189 (1989); Ra et al., Nature 241:752-754 (1989)), and one of the low affinity receptors for IgG, represented in mice by Fcxcex3RIIxcex1 (Ra et al., J. Biol. Chem. 264:15323-15327 (1989)), and in humans by the CD16 subtype expression by macrophages and natural killer cells, CD16TM (CD16 transmembrane) (Lanier et al., Nature 342:803-805 (1989); Anderson et al., Proc. Natl. Acad. Sci. USA 87:2274-2278 (1990)) and with a polypeptide of unidentified function (Anderson et al., Proc. Natl. Acad. Sci. USA 87:2274-2278 (1990)). Recently it has been reported that xcex3 is expressed by a mouse T cell line, CTL, in which it forms homodimers as well as xcex3xe2x88x92xcex6 and xcex3xe2x88x92xcex7 heterodimers (Orloff et al., Nature 347:189-191 (1990)).
The Fc receptors mediate phagocytosis of immune complexes, transcytosis, and antibody dependent cellular cytotoxicity (ADCC) (Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Unkeless et al., Annu. Rev. Immunol 6:251-281 (1988); and Mellman, Curr. Opin. Immunol. 1:16-25 (1988)). Recently it has been shown that one of the murine low affinity Fc receptor isoforms, FcRxcex3IIIB1, mediates internalization of Ig-coated targets into clathrin coated pits, and that another low affinity receptor, FcRxcex3IIIA mediates ADCC through its association with one or more members of a small family of xe2x80x98trigger moleculesxe2x80x99 (Miettinen et al., Cell 58:317-327 (1989); and Hunziker and Mellman, J. Cell Biol. 109:3291-3302 (1989)). These trigger molecules, T cell receptor (TCR) xcex6 chain, TCR xcex7 chain, and Fc receptor xcex3 chain, interact with ligand recognition domains of different immune system receptors and can autonomously initiate cellular effector programs, including cytolysis, following aggregation (Samelson et al., Cell 43:223-231 (1985); Weissman et al., Science 239:1018-1020 (1988); Jin et al., Proc. Natl. Acad. Sci. USA 87:3319-3323 (1990); Blank et al., Nature 337:187-189 (1989); Lanier et al., Nature 342:803-805 (1989); Kurosaki and Ravetch, Nature 342:805-807 (1989); Hibbs et al., Science 246:1608-1611 (1989); Anderson et al., Proc. Natl. Acad. Sci USA 87:2274-2278 (1990); and Irving and Weiss, Cell 64: 891-901 (1991)).
In drawing parallels between the murine and human low affinity Fc receptor families, however, it has become clear that the human FcRxcex3IIA and C isoforms have no murine counterpart. In part because of this, their function has yet to be defined.
Because humoral agents based on CD4 alone may have limited utility in vivo, previous work explored the possibility of augmenting cellular immunity to HIV. Preparations of protein chimeras in which the extracellular domain of CD4 is fused to the transmembrane and/or intracellular domains of T cell receptor, IgG Fc receptor, or B cell receptor signal transducing elements have been identified (U.S. Ser. Nos. 07/847,566 and 07/665,961, hereby incorporated by reference). Cytolytic T cells expressing chimeras which include an extracellular CD4 domain show potent MHC-independent destruction of cellular targets expressing HIV envelope proteins. An extremely important and novel component of this approach has been the identification of single T cell receptor, Fc receptor, and B cell receptor chains whose aggregation suffices to initiate the cellular response. One particularly useful application of this approach has been the invention of chimeras between CD4 and xcex6, xcex7, or xcex3 that direct cytolytic T lymphocytes to recognize and kill cells expressing HIV gp120 (U.S. Ser. Nos. 07/847,566 and 07/665,961, hereby incorporated by reference).
In general, the invention features a method of directing a cellular immune response against an HIV-infected cell in a mammal. The method involves administering to the mammal an effective amount of therapeutic cells, the therapeutic cells expressing a membrane-bound, proteinaceous chimeric receptor comprising (a) an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection and (b) an intracellular portion which is capable of signalling the therapeutic cell to destroy the receptor-bound HIV-infected cell.
In a related aspect, the invention features a cell which expresses a proteinaceous membrane-bound chimeric receptor which comprises (a) an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection and (b) an intracellular portion which is capable of signalling the therapeutic cell to destroy the receptor-bound HIV-infected cell.
In a second aspect, the invention features a method of treating HIV in a mammal involving administering to the mammal an effective amount of therapeutic cells, the therapeutic cells expressing a membrane-bound, proteinaceous chimeric receptor comprising an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection.
In a related aspect, the invention features a cell which expresses a membrane-bound, proteinaceous chimeric receptor comprising an extracellular portion which includes a fragment of CD4 which is capable of specifically recognizing and binding the HIV-infected cell but which does not mediate HIV infection.
In preferred embodiments of both the first and second aspects, the CD4 fragment is amino acids 1-394 of CD4 or is amino acids 1-200 of CD4; the CD4 fragment is separated from the intracellular portion by the CD7 transmembrane domain shown in FIG. 26 or by the hinge, CH2, and CH3 domains of the human IgG1 molecule shown in FIG. 25; and the CD4 fragment is separated from the therapeutic cell by at least 48 angstroms (and preferably, by at least 72 angstroms). In preferred embodiments of the first aspect, the intracellular portion is the signal-transducing portion of a T cell receptor protein (for example, xcex6), a B cell receptor protein, or an Fc receptor protein; and the therapeutic cells are selected from the group consisting of: (a) T lymphocytes; (b) cytotoxic T lymphocytes; (c) natural killer cells; (d) neutrophils; (e) granulocytes; (f) macrophages; (g) mast cells; (h) HeLa cells; and (i) embryonic stem cells (ES).
In other related aspects, the invention features DNA encoding a chimeric receptor of the invention; and a vector including that chimeric receptor DNA.
Although the specific embodiment of the present invention is a chimera between CD4 and zeta, any receptor chain having a similar function to these molecules, e.g., in granulocytes or B lymphocytes, could be used for the purposes disclosed here. The distinguishing features of a desirable immune cell trigger molecule comprises the ability to be expressed autonomously (i.e., as a single chain), the ability to be fused to an extracellular CD4 domain such that the resultant chimera is present on the surface of a therapeutic cell, and the ability to initiate cellular effector programs upon aggregation secondary to encounter with a target ligand.
At present the most convenient method for delivery of the chimeras to immune system cells is through some form of genetic therapy. However reconstituting immune system cells with chimeric receptors by mixture of the cells with suitably solubilized purified chimeric protein would also result in the formation of an engineered cell population capable of responding to HIV-infected targets. Similar approaches have been used, for example, to introduce the CD4 molecule into erythrocytes for therapeutic purposes. In this case the engineered cell population would not be capable of self renewal.
The present invention relates to functional and simplified chimeras between CD4 fragments and T cell receptor, B cell receptor, and Fc receptor subunits which are capable of directing immune cells to recognize and lyse HIV-infected cells. The method for directing the cellular response in a mammal comprises administering an effective amount of therapeutic cells (for example, cytotoxic T lymphocytes) to the mammal, the cells being capable of recognizing and destroying the HIV-infected cell.
The invention also includes the chimeric receptor proteins which direct the cytotoxic T lymphocytes to recognize and lyse HIV-infected cells, the host cells transformed with a vector comprising the chimeric receptors, and antibodies directed against the chimeric receptors.
These and other non-limiting embodiments of the present invention will be apparent to those of skill from the following detailed description of the invention.
In the following detailed description, reference will be made to various methodologies known to those of skill in the art of molecular biology and immunology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full.
Standard reference works setting forth the general principles of recombinant DNA technology include Watson et al., Molecular Biology of the Gene, volumes I and II, the Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, Calif. (1987); Darnell et al., Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, Genes II, John Wiley and Sons, publishers, New York, N.Y. (1985); Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd ed., University of California Press, publisher, Berkeley, Calif. (1981); Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, New York, N.Y. (1989).
By xe2x80x9ccloningxe2x80x9d is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
By xe2x80x9ccDNAxe2x80x9d is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a xe2x80x9ccDNA clonexe2x80x9d means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
By xe2x80x9ccDNA libraryxe2x80x9d is meant a collection of recombinant DNA molecules containing cDNA inserts which comprise DNA copies of mRNA being expressed by the cell at the time the cDNA library was made. Such a cDNA library may be prepared by methods known to those of skill, and described, for example, in Ausubel et al., supra and Maniatis et al., supra. Generally, RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purpose of the present invention are mammalian, and particularly human, lymphocytic cell lines. A presently preferred vector for this purpose is the vaccinia virus WR strain.
By xe2x80x9cvectorxe2x80x9d is meant a DNA molecule derived, e.g., from a plasmid, bacteriophage, or mammalian or insect virus, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. Thus, by xe2x80x9cDNA expression vectorxe2x80x9d is meant any autonomous element capable of directing the synthesis of a recombinant peptide. Such DNA expression vectors include bacterial plasmids and phages and mammalian and insect plasmids and viruses.
By xe2x80x9csubstantially purexe2x80x9d is meant a compound, e.g., a protein, a polypeptide, or an antibody, that is substantially free of the components that naturally accompany it. Generally, a compound is substantially pure when at least 60%, more preferably at least 75%, and most preferably at least 90% of the total material in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. In the context of a nucleic acid, xe2x80x9csubstantially purexe2x80x9d means a nucleic acid sequence, segment, or fragment that is not immediately contiguous with (i.e., covalently linked to) both of the coding sequences with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally occurring genome of the organism from which the DNA of the invention is derived.
A xe2x80x9cfragmentxe2x80x9d of a molecule, such as any of the cDNA sequences of the present invention, is meant to refer to any contiguous nucleotide subset of the molecule. An xe2x80x9canalogxe2x80x9d of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof. A molecule is said to be xe2x80x9csubstantially similarxe2x80x9d to another molecule if the sequence of amino acids in both molecules is substantially the same. In particular, a xe2x80x9csubstantially similarxe2x80x9d amino acid sequence is one that exhibits at least 50%, preferably 85%, and most preferably 95% amino acid sequence identity to the natural or reference sequence and/or one that differs from the natural or reference amino acid sequence only by conservative amino acid substitutions. Substantially similar amino acid molecules will possess a similar biological activity. As used herein, a molecule is said to be a xe2x80x9cchemical derivativexe2x80x9d of another molecule when it contains chemical moieties not normally a part of the molecule. Such moieties may improve the molecule""s solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington""s Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).
A xe2x80x9cfunctional derivativexe2x80x9d of a receptor chimera gene of the present invention is meant to include xe2x80x9cfragmentsxe2x80x9d or xe2x80x9canaloguesxe2x80x9d of the gene, which are xe2x80x9csubstantially similarxe2x80x9d in nucleotide sequence. xe2x80x9cSubstantially similarxe2x80x9d nucleic acids encode substantially similar amino acid sequences (as defined above) and also may include any nucleic acid sequence capable of hybridizing to the natural or reference nucleic acid sequence under appropriate hybridization conditions (see, for example, Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, New York, N.Y. (1989) for appropriate hybridization stringency conditions.) A xe2x80x9csubstantially similarxe2x80x9d chimeric receptor possesses similar activity to a xe2x80x9cwild-typexe2x80x9d T cell, B cell, or Fc receptor chimera. Most preferably, the derivative possesses 90%, more preferably, 70%, and preferably 40% of the wild-type receptor chimera""s activity. The activity of a functional chimeric receptor derivative includes specific binding (with its extracellular CD4 portion) to an HIV-infected cell and resultant destruction of that cell; in addition, the chimeric receptor does not render the receptor-bearing cell susceptible to HIV infection. Chimeric receptor activity may be tested using, e.g., any of the assays described herein.
A DNA sequence encoding the CD4 receptor chimera of the present invention, or its functional derivatives, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis et al., supra, and are well known in the art.
A nucleic acid molecule, such as DNA, is said to be xe2x80x9ccapable of expressingxe2x80x9d a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are xe2x80x9coperably linkedxe2x80x9d to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5xe2x80x2-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
If desired, the non-coding region 3xe2x80x2 to the gene sequence coding for the protein may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3xe2x80x2-region naturally contiguous to the DNA sequence coding for the protein, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3xe2x80x2 region functional in the host cell may be substituted.
Two DNA sequences (such as a promoter region sequence and a CD4-receptor chimera encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the receptor chimera gene sequence, or (3) interfere with the ability of the receptor chimera gene sequence to be transcribed by the promoter region sequence. A promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. Thus, to express the protein, transcriptional and translational signals recognized by an appropriate host are necessary.
The present invention encompasses the expression of a CD4-receptor chimera protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells, although eukaryotic (and, particularly, human lymphocyte) expression is preferred.
Antibodies according to the present invention may be prepared by any of a variety of methods. For example, cells expressing the CD4-receptor chimera protein, or a functional derivative thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the chimera.
In a preferred method, antibodies according to the present invention are monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-684 (1981)). In general, such procedures involve immunizing an animal with the CD4-receptor chimera antigen. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the chimera.
Antibodies according to the present invention also may be polyclonal, or, preferably, region specific polyclonal antibodies.
Antibodies against the CD4-receptor chimera according to the present invention may be used to monitor the amount of chimeric receptor (or chimeric receptor-bearing cells) in a patient. Such antibodies are well suited for use in standard immunodiagnostic assays known in the art, including such immunometric or xe2x80x9csandwichxe2x80x9d assays as the forward sandwich, reverse sandwich, and simultaneous sandwich assays. The antibodies may be used in any number of combinations as may be determined by those of skill without undue experimentation to effect immunoassays of acceptable specificity, sensitivity, and accuracy.
Standard reference works setting forth general principles of immunology include Roitt, Essential Immunology, 6th ed., Blackwell Scientific Publications, publisher, Oxford (1988); Kimball, Introduction to Immunology, 2nd ed., Macmillan Publishing Co., publisher, New York (1986); Roitt et al., Immunology, Gower Medical Publishing Ltd., publisher, London, (1985); Campbell, xe2x80x9cMonoclonal Antibody Technology,xe2x80x9d in Burdon et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, volume 13, Elsevier, publisher, Amsterdam (1984); Klein, Immunology: The Science of Self-Nonself Discrimination, John Wiley and Sons, publisher, New York (1982); and Kennett et al., eds., Monoclonal Antibodies. Hybridoma: A New Dimension In Biological Analyses, Plenum Press, publisher, New York (1980).
By xe2x80x9cdetectingxe2x80x9d it is intended to include determining the presence or absence of a substance or quantifying the amount of a substance. The term thus refers to the use of the materials, compositions, and methods of the present invention for qualitative and quantitative determinations.
The antibodies and substantially purified antigen of the present invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the assay to be used.
The types of assays which can be incorporated in kit form are many, and include, for example, competitive and non-competitive assays. Typical examples of assays which can utilize the antibodies of the invention are radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunoadsorbent assays (ELISA), and immunometric, or sandwich, immunoassays.
By the term xe2x80x9cimmunometric assayxe2x80x9d or xe2x80x9csandwich immunoassay,xe2x80x9d it is meant to include simultaneous sandwich, forward sandwich, and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
By xe2x80x9cspecifically recognizes and bindsxe2x80x9d is meant that the antibody recognizes and binds the chimeric receptor polypeptide but does not substantially recognize and bind other unrelated molecules in a sample, e.g., a biological sample.
By xe2x80x9ctherapeutic cellxe2x80x9d is meant a cell which has been transformed by a CD4-receptor chimera of the invention so that it is capable of recognizing and destroying an HIV-infected cell; preferably such therapeutic cells are cells of the hematopoietic system.
By xe2x80x9cextracellularxe2x80x9d is meant having at least a portion of the molecule exposed at the cell surface. By xe2x80x9cintracellularxe2x80x9d is meant having at least a portion of the molecule exposed to the therapeutic cell""s cytoplasm. By xe2x80x9ctransmembranexe2x80x9d is meant having at least a portion of the molecule spanning the plasma membrane. An xe2x80x9cextracellular portionxe2x80x9d, an xe2x80x9cintracellular portionxe2x80x9d, and a xe2x80x9ctransmembrane portionxe2x80x9d, as used herein, may include flanking amino acid sequences which extend into adjoining cellular compartments.
By xe2x80x9coligomerizexe2x80x9d is meant to complex with other proteins to form dimers, trimers, tetramers, or other higher order oligomers. Such oligomers may be homo-oligomers or hetero-oligomers. An xe2x80x9coligomerizing portionxe2x80x9d is that region of a molecule which directs complex (i.e., oligomer) formation.
By xe2x80x9ccytolyticxe2x80x9d is meant to be capable of destroying a cell (e.g., an HIV-infected cell) or to be capable of destroying an infective agent (e.g., an HIV virus).
By xe2x80x9cimmunodeficiency virusxe2x80x9d is meant a retrovirus that, in wild-type form, is capable of infecting T4 cells of a primate host and possesses a viral morphogenesis and morphology characteristic of the lentivirus subfamily. The term includes, without limitation, all variants of HIV and SIV, including HIV-1, HIV-2, SIVmac, SIVagm, SIVmnd, SIVsmm, SIVman, SIVmand, and SIVcpz.
By xe2x80x9cMHC-independentxe2x80x9d is meant that the cellular cytolytic response does not require the presence of an MHC class II antigen on the surface of the targeted cell.
By a xe2x80x9cfunctional cytolytic signal-transducing derivativexe2x80x9d is meant a functional derivative (as defined above) which is capable of directing at least 40%, more preferably 70%, or most preferably at least 90% of the biological activity of the wild type molecule. As used herein, a xe2x80x9cfunctional cytolytic signal-transducing derivativexe2x80x9d may act by directly signaling the therapeutic cell to destroy a receptor-bound agent or cell (e.g., in the case of an intracellular chimeric receptor portion) or may act indirectly by promoting oligomerization with cytolytic signal transducing proteins of the therapeutic cell (e.g., in the case of a transmembrane domain). Such derivatives may be tested for efficacy, e.g., using the in vitro assays described herein.
By a xe2x80x9cfunctional HIV envelope-binding derivativexe2x80x9d is meant a functional derivative (as defined above) which is capable of binding any HIV envelope protein. Functional derivatives may be identified using, e.g., the in vitro assays described herein.
The transformed cells of the present invention are used for immunodeficiency virus therapy. Current methods of administering such transformed cells involve adoptive immunotherapy or cell-transfer therapy. These methods allow the return of the transformed immune-system cells to the bloodstream. Rosenberg, Sci. Am. 62 (May 1990); Rosenberg et al., N. Engl. J. Med. 323:570 (1990).
The pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.
The drawings will first be described.